Preventives and/or remedies for subjects with the expression or activation of her2 and/or egfr

ABSTRACT

A Her2 and/or EGFR inhibitor to be administered to a subject determined to show overexpression or activation of Her2 and/or EGFR as a result of a diagnosis of the subject for the expression or activity of Her2 and/or EGFR based on a test for detecting the expression or activity of Her2 and/or EGFR, and a pharmaceutical composition containing the inhibitor.

TECHNICAL FIELD

The present invention relates to a pharmaceutical agent to be used for aseries of treatment methods comprising diagnosing a subject forexpression or activation of Her2 and/or EGFR and treating the subjectconfirmed to show expression thereof.

BACKGROUND ART

Conventionally, the target molecules of anti-cancer agents have beenmainly those related with DNA or RNA synthesis and cell division. Suchanti-cancer agents are known to cause severe side effects such as bonemarrow toxicity and the like, because the target molecules are notspecific for cancer cells.

With the development of molecular oncology in recent years, it has beenclarified that cancer is induced by abnormality of oncogenes or tumorsuppressor genes. As the most well known oncogenes, epithelial growthfactor receptor (hereinafter to be abbreviated as EGFR) and analogoushuman EGFR type 2 (hereinafter to be abbreviated as Her2, differentname, also referred to as erbB-2) can be mentioned.

EGFR and Her2 are both transmembrane glycoproteins respectively having amolecular weight of 170 kD, 185 kD. A tyrosine kinase domain is presentin the intracellular domains of EGFR and Her2, and transduct signals tothe nucleus via phosphorylation reactions. The amino acid sequences ofkinase domain have about 80% of homology and are structurally highlysimilar. However, the homology of autophosphorylation domain in theC-terminal is as low as about 30%, which suggests qualitative differencein the signal transduction mechanisms of the both receptors.

EGFR expresses in the esophageal cancer at an extremely high frequencyof 89% [J. Cancer Res. Clin. Oncol. 1993 119, 401-407]. In othercancers, it has been reported that an overexpression of EGFR is found in45% of non-small cell lung cancer [Cancer Res. 1993 53, 2379-2385], geneamplification of EGFR in 50% of glioblastoma [Cancer Res. 2000 60,1383-1387] and the like.

As for Her2, gene amplification is observed in 39% of breast cancer [J.Clin. Oncol. 1993 11, 1936-1942], overexpression is observed in 32% ofovarian cancer [Cancer Res. 1990 50, 4087-4091], overexpression isobserved in 67% of hormone-resistant prostate cancer [Clinical CancerRes. 2001 7, 2643-2647] and the like.

While EGFR and Her2 are over-expressed in various cancers, thesubstantial molecular mechanism of tumorigenesis by them is a signaltransduction via a phosphorylation reaction, and the extensiveactivation of signal transduction may clinically deteriorate cancerprogression independently of the level of expression amount of EGFR andHer2. Therefore, as long as the target expression varies depending onthe cancers, a treatment targeting these molecules require individualtreatments, wherein the nature of the cancer should be diagnosed, and apharmaceutical agent should be determined depending on the presence orabsence of expression and activation of the target molecules. Thisaffords an effective and reasonable treatment.

EGFR and Her2 form a heterocomplex and shows functional interaction [J.Clin. Oncol. 2001 19(18s), 32s-40s]. It is known that coexpression ofEGFR and Her2 accelerates tumorigenesis by EGFR alone [Cell 1987 58,287-292]. In addition, there are reports that coexpression of EGFR andHer2 in breast cancer, oral cavity cancer, lung cancer and the likeleads to poor prognosis [Clin. Cancer Res. 1999 5, 4164-4174].

Therefore, a pharmaceutical agent inhibiting both EGFR and Her2(hereinafter also referred to simply as a dual inhibitor) isadvantageous in that it is applicable to a wider range of diseases ascompared to a pharmaceutical agent acting only on either of them, andalso superior in that it has a potential to provide an effectivetreatment of coexpressed cancers based on a synergistic action of dualinhibition.

The diagnostic techniques of EGFR and Her2 are already widely known. Arecent report has documented on an attempt to detect a precancer lesionof oral cavity cancer by conjugating a fluorescence dye to an antibodybinding to EGFR [Cancer Res. 2061 61, 4490-4496]. In addition, a reporthas also appeared that suggests that an antitumor effect onanthracycline anti-cancer agents can be predicted by the presence orabsence of Her2 expression in breast cancer patients [Clin. Cancer Res.2001 7, 1577-1581]. Therefore, diagnosis of EGFR and Her2 may be usefulfor both the early discovery of disease and determination ofpharmaceutical agents effective and suitable for individual patients.

As an EGFR inhibitor, neutralizing antibody and tyrosine kinaseinhibitor are in the stage of clinical development for anti-canceragents. As Her2 inhibitor, a neutralizing antibody Herceptin (productname, Roche) has been already marketed in various countries as atherapeutic agent for metastatic breast cancer. However, tyrosine kinaseinhibitor is in the stage of clinical development for anti-cancer agent.

Similarly, a dual inhibitor of EGFR and Her2 has been studied anddeveloped but has not been put to practical use.

The anti-Her2 antibody Herceptin has been already subjected to thepatient selection with Her2 expression using diagnosis Herceptest(product name). Its indication is limited to breast cancer.

An EGFR kinase inhibitor Iressa (product name, A. Zeneca) has beenproven clinically to cause no severe side effects that conventionalanti-cancer agents have, such as bone marrow toxicity and the like, andto be effective for non-small cell lung cancer [Clin. Cancer Res. 20017, 3780s]. In clinical studies for Iressa, however, prior confirmationof EGFR expression was not performed for selection of patients.

A dual inhibitor of EGFR and Her2 can be applied to a wide range ofcancers, because it can be applied as long as either target molecule isexpressed therein. As mentioned earlier, however, prescription forpatients, which are appropriately determined based on the diagnosis ofexpression or activation of target protein, is desirable in a moreprecise sense, and this series of treatment methods using a dualinhibitor has not been established yet in clinical situations.

As a different example relating to the expression of target molecule incancer tissues and the sensitivity of anti-cancer agents, a report hasdocumented that the expression amount of thymidylate synthase (TS) anddihydropyrimidine dehydrogenase activity in breast cancer tissues, and 5FU sensitivity are related [Journal of Japan Society of ClinicalOncology, 2000 35, 340]. However, TS expression diagnosis has not yetled to be performed in fact as a treatment method before prescription of5 FU.

As mentioned above, it has been desired to predict an effectivetreatment method by diagnosing the target molecules of thepharmaceutical agents for prescription of anti-cancer agents.

DISCLOSURE OF THE INVENTION

In view of the above-mentioned situation, the present inventors haveconducted intensive studies in an attempt to solve the aforementionedproblems and found that a series of treatment methods can be establishedby diagnosing a subject for expression or activity of Her2 and/or EGFR,and administering a Her2 and/or EGFR inhibitor to the subject confirmedto show the expression or activity.

Namely, the present invention relates to a Her2 and/or EGFR inhibitor tobe administered in such series of treatment methods, a pharmaceuticalcomposition containing the inhibitor as an active ingredient, and aprophylactic and/or therapeutic method comprising administering theinhibitor to a subject, and more particularly, the present inventionprovides the following.

The present invention relates to

-   (1) a Her2 and/or EGFR inhibitor to be administered to a subject    determined to show overexpression or activation of Her2 and/or EGFR    as a result of a diagnosis of the subject for the expression or    activity of Her2 and/or EGFR based on a test for detecting the    expression or activity of Her2 and/or EGFR,-   (2) the inhibitor of the aforementioned (1) to be administered to a    subject determined to show activation of Her2 and/or EGFR as a    result of a diagnosis of the subject for the activity of Her2 and/or    EGFR based on a test for detecting the activity of Her2 and/or EGFR,-   (3) the inhibitor of the aforementioned (1), which is a Her2 and    EGFR inhibitor for administration to a subject determined to show    overexpression or activation of Her2 and EGFR as a result of a    diagnosis of the subject for the expression or activity of Her2 and    EGFR based on a test for detecting the expression or activity of    Her2 and EGFR,-   (4) the inhibitor of the aforementioned (3) to be administered to a    subject determined to show activation of Her2 and EGFR as a result    of a diagnosis of the subject for the activity of Her2 and EGFR    based on a test for detecting the activity of Her2 and EGFR,-   (5) the inhibitor of the aforementioned (1), wherein the subject is    expected to suffer from a disease caused by overexpression or    activation of Her2 and/or EGFR,-   (6) the inhibitor of the aforementioned (3), wherein the subject is    expected to suffer from a disease caused by overexpression or    activation of Her2 and EGFR,-   (7) the inhibitor of any of the aforementioned (1) to (6), wherein    the subject is a human,-   (8) the inhibitor of the aforementioned (1), wherein the test for    detecting the expression or activity of Her2 and/or EGFR is an    extracorporeal test,-   (9) the inhibitor of the aforementioned (3), wherein the test for    detecting the expression or activity of Her2 and EGFR is an    extracorporeal test,-   (10) the inhibitor of the aforementioned (3), which is a mixture of    a Her2 inhibitor and an EGFR inhibitor,-   (11) the inhibitor of the aforementioned (3), which is used for    administering a Her2 inhibitor and/or an EGFR inhibitor    simultaneously, separately or at time intervals,-   (12) the inhibitor of the aforementioned (8) or (9), wherein the    extracorporeal test is an immunological method using an antibody, or    a hybridization method using a nucleic acid and a nucleic acid    derivative,-   (13) the inhibitor of the aforementioned (12), wherein the    immunological method using an antibody is selected from the group    consisting of an enzyme-linked immunosorbent assay, an enzyme-linked    immunoassay, a radioimmunoassay, an immunohistochemical method and    western blotting,-   (14) the inhibitor of the aforementioned (12), wherein the    hybridization method using a nucleic acid and a nucleic acid    derivative is selected from the group consisting of an RT-PCR    method, an ISH method, a FISH method, northern blotting and southern    blotting method,-   (15) the inhibitor of any of the aforementioned (1) to (14), which    is a substituted heteroaromatic compound represented by the    following formula (I)    wherein X is N or CH; Y is CR¹ and V is N; or Y is N and V is CR¹;    or Y is CR¹ and V is CR²; or Y is CR² and V is CR¹; R¹ is C₁₋₄    alkyl, C₁₋₄ alkoxy, CH₃SO₂CH₂CH₂NHCH₂—Ar— (wherein Ar is selected    from phenyl, furan, thiophene, pyrrole and thiazole, each of which    is optionally substituted by 1 or 2 halogens, C₁₋₄ alkyl or C₁₋₄    alkoxy on demand) or —C═C—C(R⁶) (R⁷) (R⁸) (wherein R⁶, R⁷ and R⁸ are    each independently a hydrogen atom, hydroxy, halogen, C₁₋₄ alkyl or    C₁₋₄ alkoxy, or C₃₋₆ cycloalkyl wherein the ring is optionally    substituted by hydrogen atom or C₁₋₄ alkyl and optionally contains 1    or 2 hetero atoms selected from O, S and N therein; R² is selected    from the group consisting of hydrogen, halogen, hydroxy, C₁₋₄ alkyl,    C₁₋₄ alkoxy, C₁₋₄ alkylamino, di[C₁₋₄ alkyl]amino and —NHCO—R⁹    (wherein R⁹ is C₁₋₄ alkyl, C₁₋₄ alkoxy, C₂₋₄ alkenyl or C₂₋₄    alkynyl); U is phenyl, pyridyl, 3H-imidazolyl, indolyl, isoindolyl,    indolinyl, isoindolinyl, 1H-indazolyl, 2,3-dihydro-1H-indazolyl,    1H-benzimidazolyl, 2,3-dihydro-1H-benzimidazolyl or    1H-benzotriazolyl group, each of which is substituted by R³ group    and optionally substituted on demand by at least one R⁴ group    selected independently; R³ is selected from the group consisting of    benzyl, halo-, dihalo- and trihalobenzyl, benzoyl, pyridylmethyl,    pyridylmethoxy, phenoxy, benzyloxy, halo-, dihalo- and tribenzyloxy    and benzenesulfonyl; or R³ is trihalomethylbenzyl or    trihalomethylbenzyloxy; or R³ is a group of the above-mentioned    formula (a) (wherein each R⁵ is independently selected from halogen,    C₁₋₄ alkyl and C₁₋₄ alkoxy; and n is 0-3); each R⁴ is independently    hydroxy, halogen, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄    alkoxy, amino, C₁₋₄ alkylamino, di[C₁₋₄ alkyl]amino, C₁₋₄ alkylthio,    C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ alkylcarbonyl, carboxy,    carbamoyl, C₁₋₄ alkoxycarbonyl, C₁₋₄ alkanoylamino, N-(C₁₋₄    alkyl)carbamoyl, N,N-di(C₁₋₄ alkyl)carbamoyl, cyano, nitro or    trifluoromethyl, or a pharmaceutically acceptable salt thereof, a    hydrate or solvate thereof, an optically active substance or a    racemate thereof, or a mixture of diastereomers thereof,-   (16) the inhibitor of any of the aforementioned (1) to (15), which    is    (4-(3-fluorobenzyloxy)-phenyl)-(6-(5-((2-methanesulfonyl-ethylamino)methyl)-furan-2-yl)-pyrido[3,4-d]pyrimidin-4-yl)-amine;-   (4-benzyloxyphenyl)-(6-(5-((2-methanesulfonyl-ethylamino)-methyl)-furan-2-yl)-quinazolin-4-yl)-amine;-   N-{4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine;-   N-[4-(benzyloxy)phenyl]-7-methoxy-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine;-   N-(1-benzyl-1H-indazol-5-yl)-7-methoxy-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine;-   N-{3-fluoro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine;-   N-[1-(3-fluorobenzyl)-1H-indazol-5-yl]-6-[2-({[2-(methylsulfonyl)ethyl]amino}methyl)-1,3-thiazol-4-yl]-4-quinazolinamine;-   6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-N-[4-(phenylsulfonyl)phenyl]-4-quinazolinamine;-   N-{3-fluoro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[2-({[2-(methylsulfonyl)ethyl]amino}methyl)-1,3-thiazol-4-yl]-4-quinazolinamine;-   N-(1-benzyl-1H-indazol-5-yl)-6-[2-({[2-(methylsulfonyl)ethyl]amino}methyl)-1,3-thiazol-4-yl]-4-quinazolinamine;-   N-(3-fluoro-4-benzyloxyphenyl)-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-4-furyl]-4-quinazolinamine;-   N-(3-chloro-4-benzyloxyphenyl)-6-[2-({[2-(methylsulfonyl)ethyl]amino}methyl)-4-furyl]-4-quinazolinamine;-   N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine;-   N-(1-benzyl-1H-indazol-5-yl)-7-fluoro-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine;-   N-(3-trifluoromethyl-4-benzyloxyphenyl)-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-4-furyl]-4-quinazolinamine;-   N-[4-(3-chloro-4-fluorophenyl)amino-7-[3-(4-morpholinyl)propoxy]quinazolin-6-yl]acrylamide;-   N-{4-[(3-chloro-4-fluorophenyl)amino]-7-[3-methyl-3-(4-methyl-1-piperazinyl)-1-butynyl]-6-quinazolinyl}acrylamide;    or-   N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine,    or a pharmaceutically acceptable salt thereof, a hydrate or a    solvate thereof, an optically active substance or a racemate    thereof, or a mixture of diastereomers thereof,-   (17) the inhibitor of any of the aforementioned (1) to (16), which    is    N-[4-(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]quinazolin-6-yl]acrylamide,    or N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-5    (methanesulfonyl)ethyl]amino)methyl)-2-furyl]-4-quinazolinamine or a    pharmaceutically acceptable salt thereof, a hydrate or a solvate    thereof, an optically active substance or a racemate thereof, or a    mixture of diastereomers thereof,-   (18) a pharmaceutical composition comprising an inhibitor of any of    the aforementioned (1) to (17) as an active ingredient and a    pharmaceutically acceptable carrier,-   (19) the pharmaceutical composition of the aforementioned (18),    which is an agent for the prophylaxis and/or treatment of a disease    caused by overexpression or activation of Her2 and/or EGFR,-   (20) the pharmaceutical composition of the aforementioned (19),    wherein the disease caused by the overexpression or activation of    Her2 and/or EGFR is cancer, angiogenesis associated with the growth    of cancer or sarcoma, angiogenesis associated with cancer    metastasis, angiogenesis associated with diabetic retinopathy,    arteriosclerosis or psoriasis,-   (21) an agent for the prophylaxis and/or treatment of a disease    caused by overexpression or activation of Her2 and/or EGFR, which is    to be administered to a subject determined to show overexpression or    activation of Her2 and/or EGFR as a result of a diagnosis of the    subject for the expression or activity of Her2 and/or EGFR based on    a test for detecting the expression or activity of Her2 and/or EGFR,-   (22) the agent of the aforementioned (21), wherein the disease    caused by overexpression or activation of Her2 and/or EGFR is    cancer, angiogenesis associated with the growth of cancer or    sarcoma, angiogenesis associated with cancer metastasis,    angiogenesis associated with diabetic retinopathy, arteriosclerosis    or psoriasis,-   (23) a method for the prophylaxis and/or treatment of a disease    caused by overexpression or activation of Her2 and/or EGFR, which    comprises administering an effective dose of a Her2 and/or an EGFR    inhibitor to a subject determined to show overexpression or    activation of Her2 and/or EGFR as a result of a diagnosis of the    subject for the expression or activity of Her2 and/or EGFR based on    a test for detecting the expression or activity of Her2 and/or EGFR,-   (24) the method of the aforementioned (23), wherein the disease    caused by overexpression or activation of Her2 and/or EGFR is    cancer, angiogenesis associated with the growth of cancer or    sarcoma, angiogenesis associated with cancer metastasis,    angiogenesis associated with diabetic retinopathy, arteriosclerosis    or psoriasis,-   (25) a commercial package comprising the pharmaceutical composition    of any of the aforementioned (18) to (20) and a written matter    associated therewith, the written matter stating that the    pharmaceutical composition can or should be used for the prophylaxis    and/or treatment of a disease caused by overexpression or activation    of Her2 and/or EGFR, and-   (26) the commercial package of the aforementioned (25), wherein the    disease caused by overexpression or activation of Her2 and/or EGFR    is cancer, angiogenesis associated with the growth of cancer or    sarcoma, angiogenesis associated with cancer metastasis,    angiogenesis associated with diabetic retinopathy, arteriosclerosis    or psoriasis.

The respective definitions in the present invention are as follows.

As the “test for detecting expression of Her2 and/or EGFR”, animmunological method using an antibody against Her2 and/or EGFR or ahybridization method using a nucleic acid and a nucleic acid derivativecan be mentioned. More preferable specific examples of the immunologicalmethod include an enzyme-linked immunosorbent assay, an enzyme-linkedimmunoassay, a radioimmunoassay, an immunohistochemical method, westernblotting and the like, and more preferable specific examples of thehybridization method include an RT-PCR method, an ISH method, a FISHmethod, northern blotting, southern blotting and the like.

The “enzyme-linked immunosorbent assay (ELISA)” is an enzyme-linkedimmunoassay (EIA) performed on a solid phase, which comprises labelingan antigen or antibody with an enzyme via a covalent bond and performingan enzyme-linked immunoassay for detecting the presence, on a solidphase, of the antibody or antigen utilizing the enzyme activity. Thismethod is a radioimmunoassay (RIA) developed by E. Engval et al. in1971, except that a radioisotope with which to label either antigen orantibody is replaced by a nonradioactive enzyme. In 1990, it wasdeveloped as a method also capable of measuring an antigen of azeptomole (10⁻²¹ mol) level.

The ELISA method includes direct antibody method, indirect antibodymethod, competitive method, two antibody sandwich method and the like,and a method suitable for assay object is selected. As the solid phase,agarose, microtiter well, latex particles and the like are used, as thelabeling enzyme, peroxidase derived from horseradish is most frequentlyused. As other enzymes, alkaliphosphatase, galactosidase and the likeare also used. P In the present invention, an immunohistochemical methodis also applicable.

“Western blotting” is a method for detecting a protein transferred on amembrane such as PVDF membrane and the like, using an antibody. Since aspecific bond between antibody and antigen is utilized, only a smallamount of a sample is required and a specific target protein can bedetected.

By the “hybridization” is meant interaction between complementarynucleic acid strands. Since DNA has a double stranded structure based onthe complementary interaction (C is always bonded to G and A is alwaysbonded to T), when the complementary strands are separated, theypreferably reanneal, or “hybridize” with each other. This also occursbetween two DNA strands and between a DNA strand and an RNA strand,having sufficiently complementary base sequence. Hybridization occurs inall physiological reactions of DNA, such as replication, transcriptionand the like, and forms a basis of many molecular biological methodssuch as southern blotting, northern blotting, PCR and the like.

“PCR (Polymerase Chain Reaction)” means a reaction for specificallyamplifying a DNA fragment sandwiched between one set of primers byperforming DNA polymerase reactions continuously and as chain reactions.Of these, the “RT-PCR (reverse transcription-polymerase chain reaction)method” combining a reverse transcription reaction and PCR shows highestsensitivity.

The “ISH (in situ hybridization) method” is an effective means as adetection method of gene expression in tissue fragment. The FISH methodis a combination of this method and a fluorescence detection method.

The “northern blotting” is a technique aiming at analysis of mRNA. Itcomprises electrophoresis of RNA, which easily takes a secondarystructure, under degenerative conditions, and transfer thereof onto amembrane (nylon, nitrocellulose etc.). According to the degenerativemethod, there are 1. a method using formamide and formaldehyde, 2. amethod using gyloxal, 3. a method using methylmercury hydroxide and thelike.

The “southern blotting” is a method of transfer described by Southern in1975, wherein a transcribed DNA region having a base sequencecomplementary to a labeled nucleic acid probe is detected.

For detection of expression or activity of Her2 and/or EGFR, a tissue(cancer tissue, blood vessel wall tissue, skin, oral mucosa etc.) or abody fluid (blood, lymph) and the like, which is obtained from patients,is applied to a test as recited in the above to detect expression oractivity of Her2 and/or EGFR.

Specific examples of “a disease caused by overexpression or activationof Her2 and/or EGFR” include cancers such as brain tumor, pharyngealcancer, laryngeal cancer, tongue cancer, esophageal cancer, gastriccancer, colorectal cancer, non-small cell lung cancer, pancreaticcancer, bile duct cancer, gallbladder cancer, liver cancer, renalcancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer,cervical cancer, endometrial cancer, skin cancer, childhood solidcancer, bone tumor, hemangioma and the like, angiogenesis associatedwith diabetic retinopathy, arteriosclerosis, psoriasis and the like.Preferred are brain tumor, pharyngeal cancer, laryngeal cancer, tonguecancer, esophageal cancer, gastric cancer, colorectal cancer, non-smallcell lung cancer, pancreatic cancer, bile duct cancer, gallbladdercancer, liver cancer, renal cancer, bladder cancer, prostate cancer,breast cancer, ovarian cancer, cervical cancer, endometrial cancer, skincancer and the like and more preferred are brain tumor, gastric cancer,colorectal cancer, non-small cell lung cancer, pancreatic cancer, renalcancer, prostate cancer, breast cancer, ovarian cancer and the like.

The “overexpression or activation of Her2 and/or EGFR” is an expressionor activity not less than the expression amount or activity necessaryfor homeostasis of living organisms, and the expression or activity notless than the expression amount or activity necessary for normal tissueof the same origin.

The “patients showing overexpression or activation of Her2 and/or EGFR”means the patients wherein at least one of Her2 and EGFR is excessivelyexpressed or activated, and preferably the patients wherein both areexcessively expressed or activated. The Her2 and/or EGFR inhibitor ofthe present invention is characterized by administration for thetreatment of patients, wherein Her2 and/or EGFR are/is excessivelyexpressed or activated as mentioned above.

The “Her2 and/or EGFR inhibitor” of the present invention is preferablya Her2 and EGFR inhibitor to be administered to patients wherein Her2and EGFR are excessively expressed or activated, and may be a mixture ofa Her2 inhibitor and an EGFR inhibitor. It is possible to use a Her2inhibitor and an EGFR inhibitor simultaneously, separately or at timeintervals. In other words, it is possible to administer a Her2 inhibitorand an EGFR inhibitor simultaneously, separately or, for example, in astaggered manner in a single day or at given time intervals for severaldays to several weeks or several months, by various different routes.

The Her2 inhibitor to be used in the present invention includes ananti-Her2 antibody Herceptin (Roche), TAK-165 (Takeda), ETH-102 (ExonHitTher.) and the like, and the EGFR inhibitor to be used in the presentinvention includes Iressa (A. Zeneca), OSI-774 (Roche), PKI-166(Novartis), EKB-569 (Wyeth) and the like. The production thereof and thelike are described in WO02/06249, JP-A-2001-348385, JP-A-2002-69070,JP-A-9-136877, JP-A-11-60571 and the like for TAK-165, WO96/33980 andpatent No. 3040486 for Iressa, WO96/30347 for OSI-774, WO97/02266 forPKI-166, and U.S. Pat. No. 6,002,008 for EKB-569.

When these are prepared into a mixture of a Her2 inhibitor and an EGFRinhibitor, one or more of respective inhibitors are selected and amixture thereof is produced by an appropriate known method and put touse.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chemiluminescence taken with a luminoCCD camera inExample 4.

BEST MODE FOR EMBODYING THE INVENTION

As the inhibitor of the present invention, a dual inhibitor ispreferable. Specific examples thereof include an inhibitor that actsbased on inhibition of the protein kinase activity by the enzyme, aninhibitor that acts by decreasing the Her2 and/or EGFR intracellularprotein content, or an inhibitor of a physical interaction between Her2and/or EGFR and a signal transduction molecule, and the like.

As examples of more specific compound of the “dual inhibitor”, asubstituted heteroaromatic compound represented by the following formula(I), which is disclosed in WO99/35146 (JP-T-2002-500225)

wherein X is N or CH; Y is CR¹ and V is N; or Y is N and V is CR¹; or Yis CR¹ and V is CR²; or Y is CR² and V is CR; R¹ isCH₃SO₂CH₂CH₂NHCH₂—Ar— group (wherein Ar is selected from phenyl, furan,thiophene, pyrrole and thiazole, each of which is optionally substitutedby 1 or 2 halogens, C₁₋₄ alkyl or C₁₋₄ alkoxy group on demand); R² isselected from the group consisting of hydrogen, halogen, hydroxy, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylamino and di[C₁₋₄ alkyl]amino; U isphenyl, pyridyl, 3H-imidazolyl, indolyl, isoindolyl, indolinyl,isoindolinyl, 1H-indazolyl, 2,3-dihydro-1H-indazolyl, 1H-benzimidazolyl,2,3-dihydro-1H-benzimidazolyl or 1H-benzotriazolyl group, each of whichis substituted by R³ group and optionally substituted on demand by atleast one R⁴ group independently selected; R³ is selected from the groupconsisting of benzyl, halo-, dihalo- and trihalobenzyl, benzoyl,pyridylmethyl, pyridylmethoxy, phenoxy, benzyloxy, halo-, dihalo- andtribenzyloxy and benzenesulfonyl; or R³ is trihalomethylbenzyl ortrihalomethylbenzyloxy; or R³ is a group of the above-mentioned formula(a) (wherein each R⁵ is independently selected from halogen, C₁₋₄ alkyland C₁₋₄ alkoxy; and n is 0-3); each R⁴ is independently hydroxy,halogen, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ alkoxy, amino,C₁₋₄ alkylamino, di[C₁₋₄ alkyl]amino, C₁₋₄ alkylthio, C₁₋₄alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ alkylcarbonyl, carboxy,carbamoyl, C₁₋₄ alkoxycarbonyl, C₁₋₄ alkanoylamino, N-(C₁₋₄ alkyl)carbamoyl, N,N-di (C₁₋₄ alkyl)carbamoyl, cyano, nitro ortrifluoromethyl, particularly preferably,N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamineand the like, orN-[4-(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]quinazolin-6-yl]acrylamideand the like disclosed in WO00/31048 can be mentioned.

The compound of the formula (I) may take any form of a pharmaceuticallyacceptable salt, a hydrate or a solvate, a geometric isomer, an opticalisomer or a racemate or a diastereomer mixture. As the pharmaceuticallyacceptable salt, salts with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid andsulfuric acid and the like, salts with organic acids such as formicacid, acetic acid, tartaric acid, trifluoroacetic acid, citric acid,malic acid, lactic acid, fumaric acid, maleic acid, benzoic acid,phthalic acid, glycolic acid, glucuronic acid, gluconic acid, succinicacid, methanesulfonic acid, p-toluenesulfonic acid, isethionic acid andthe like, salts with alkali metals or alkaline earth metals such assodium, potassium, magnesium, calcium and the like, salts with organicbases such as ammonium, tetramethylamine, triethylamine, benzylamine,phenethylamine, monoethanolamine, diethanolamine,tris(hydroxyethylamine) and the like, salts with amino acids such aslysin, arginine, aspartic acid, glutamic acid and the like can bementioned.

The production method and dual inhibitory action of the compound of theformula (I) are described in WO99/35146, and the production method anddual inhibitory action ofN-[4-(3-chloro-4-fluorophenyl)amino-7-[3-(4-morpholinyl)propoxy]quinazolin-6-yl]acrylamideare described in WO00/31048.

The dual inhibitor of the present invention contains any of theabove-mentioned compounds and other compounds having a dual inhibitoryaction (e.g., compound described in WO02/066451).

When the inhibitor of the present invention is used as a pharmaceuticalagent, the inhibitor of the present invention is mixed with apharmaceutically acceptable carrier (excipient, binder, disintegrant,corrigent, flavor, emulsifier, diluent, dissolution aids etc.) and canbe administered orally or parenterally in the form of the obtainedpharmaceutical composition or preparation (tablet, pill, capsule,granule, powder, syrup, emulsion, elixir, suspension, solution,injection, infusion, suppository etc.).

In the present invention, parenteral includes subcutaneous injection,intravenous injection, intramuscular injection, intraperitonealinjection, infusion method and the like.

As a dosage form of a solid administration for oral administration,those mentioned above such as powder, granule, tablet, pill, capsule andthe like can be mentioned. In such dosage form, the active ingredientcompound can contain at least one pharmaceutically acceptable additive(inert diluent, lubricant, preservative, antioxidant, disintegrant,binder, thickener, buffer, sweetener, flavor, perfume etc.).

As the liquid for oral administration, pharmaceutically acceptableemulsion, syrup, elixir, suspension, solution and the like can bementioned, which may contain an inert diluent usually employed in thepertinent field.

A preparation for injection (sterile aqueous suspension or oilsuspension for injection etc.) can be prepared by a method known in thepertinent field and using a suitable dispersing agent, wetting agent,suspending agent and the like. The sterile preparation for injection maybe a sterile injectable solution or suspension using a diluent orsolvent. In addition, a sterile involatile oil can be generally used asa solvent or suspending solvent. For this end, any involatile oil orfatty acid can be used.

The suppository for rectal administration can be produced by mixing thedrug and a suitable non-stimulant excipient, such as one that is solidat ordinary temperature, liquid at the temperature of intestine, andmelts in rectal to release the drug, and the like.

The dose is determined in consideration of the age, body weight, generalhealth conditions, sex, diet, administration time, administrationmethod, excretion speed, combination of drugs, level of disease forwhich the patient is under treatment or other factors. For example, whenthe above-mentioned compound represented by the formula (I) is used, thedaily dose thereof varies depending on the condition and body weight ofthe patients, kind of the compound, administration route and the like.The compound is orally administered in a dose of 0.01-1000 mg/kg bodyweight/day, preferably 0.05-500 mg/kg body weight/day, which is once toseveral times a day. Parenterally, about 0.01-50 mg/kg body weight/day,preferably 0.01-20 mg/kg body weight/day, is preferably administeredsubcutaneously, intravenously, intramuscularly or rectally.

The prophylactic and/or therapeutic agent of the present invention to beused for patients who showed expression or activation of Her2 and/orEGFR is preferably provided as a pharmaceutical product by packagingwith a written matter stating that the agent can or should be used forthe patients determined to be suffering from a disease caused byexpression or activation of Her2 and/or EGFR as a result of a diagnosisof the presence of expression or activity of Her2 and/or EGFR based on atest for detecting the expression or activity of Her2 and/or EGFR.

EXAMPLES

The present invention is explained in more detail by illustratingExamples in the following, which are not to be construed as limitativeunless they go beyond the gist of the invention.

Example 1 EGFR Tyrosine Kinase Inhibitory Activity

(Method)

The pharmaceutical agent PD 0183805 used for the test is known toinhibit EGFR tyrosine kinase and shows an in vivo antitumor effect onEGFR overexpression cancer A431¹). In addition, PD 0183805dihydrochloride CI-1033 has been reported to inhibit tyrosinephosphorylation of Her2, erbB3 and erbB4 when MDA-MB-453 cells arestimulated with Heregulin²).

In the following description, PD 0183805 is abbreviated as PD, and PD0183805 dihydrochloride CI-1033 is abbreviated as PD.2HCl. The chemicalname and chemical structure of PD is as follows.

-   N-[4-(3-chloro-4-fluorophenyl)amino-7-[3-(4-morpholinyl)propoxy]quinazolin-6-yl]acrylamide

In addition, PD and PD.2HCl was synthesized according to the methoddescribed in WO00/31048.

-   1) Vincent, P. W., Patmore, S. J., Bridges, A. J., Kirkish, L. S.,    Dudeck, R. C., Leopold, W. R., Zhou, H., Elliott, W. L. Proc. Am.    Assoc. Cancer Res., 40: 117, 1999.-   2) Slichenmyer, W. J., Elliott, W. L. and Fry, D. W. Semin. Oncol.,    28: 80, 2001.

The test was performed using partially purified EGFR obtained from humanepidermoid cancer cell A431 (provided by Cell Resource Center forBiomedical Research, the Institute of Development, Aging and Cancer,Tohoku University; catalog No. TKG-0182, or when purchased from ATCC;ATCC No. CRL-1555) according to a modification of the tyrosine kinaseactivity assay method of Linda J. Pike et al. (Proceedings of theNational Academy of Science of the U.S.A., 1982, 79, 1433). The methodwas detailedly as follows.

A431 cells were incubated in a 10% FBS supplemented DMEM medium at 37°C. under 5% carbon dioxide, and the cells were homogenized in a solutioncontaining 10 mM HEPES buffer (pH 7.4), 0.25 M sucrose and 0.1 mM EDTA,after which centrifuged at 3,000×G for 5 min. The supernatant wascentrifuged at 100,000×G for 30 min to give an A431 cell membranefraction, which was subjected to the assay as the partially purifiedEGFR as an enzyme source.

To a reaction mixture (final concentration 1% DMSO) of thepharmaceutical agent dissolved in dimethyl sulfoxide (DMSO), the A431cell membrane fraction (10-15 μg), 30 mM HEPES buffer (pH 7.4), 2 mMMnCl₂ and 100 μM Na₃VO₄ was added 100 ng of EGF and synthetic substrateangiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, 50 μg) and adenosinetriphosphate (containing 37 KBq of γ-³²P-labeled form, finalconcentration 10 μM) were further added to start the reaction. Thevolume then was 60 μL.

The reaction was carried out in ice for 30 min and stopped by theaddition of 10 mg/mL bovine serum albumin (6 μL) and 20% trichloroaceticacid (25 μL). The reaction mixture was left standing in the ice for 30min.

After centrifugation at 5000G for 2 min, the supernatant was sampled (40μL) and allowed to adsorb onto P81 phosphocellulose paper. These wereimmersed in 0.75% aqueous phosphoric acid for 5 min for washing and thiswashing operation was repeated 4 times. The paper was taken out andmeasured for ³²P count on a liquid scintillation counter, and theobtained value was taken as A.

A reaction without a pharmaceutical agent and a reaction without both apharmaceutical agent and EGF were simultaneously measured for ³²P count,and the obtained values were taken as B and C, respectively.

From these values, the tyrosine kinase inhibitory rate can be determinedfrom the following formula:Inhibitory rate (%)=100−{(A−C)/(B−C)}×100

IC₅₀ (50% inhibitory concentration) was calculated from the inhibitoryrates obtained by changing the addition concentration of thepharmaceutical agent. The results are shown in the following table.TABLE 1 EGFR tyrosine kinase inhibitory activity pharmaceutical agentIC₅₀ nM PD 0.4

Example 2 Cellular Her2 Tyrosine Kinase Inhibitory activity

(Method)

As the cells, NIH3T3 mouse fibroblast cell line (provided by CellResource Center for Biomedical Research, the Institute of Development,Aging and Cancer, Tohoku University; catalog No. TKG-0298) transformedwith mutant c-erbB2 constitutively activated by substituting 659thvaline by glutamic acid (hereinafter to be referred to as A4 cell) wasused. This cell line was cultured and maintained in a 10% FBSsupplemented DMEM/F12 mixed medium (hereinafter to be referred to as anassay medium) in a plastic dish at 37° C. under 5% carbon dioxide.

A4 cells suspended in an assay medium were seeded in a 12 well plate at3×10⁵/well, and the cells that reached confluent were incubated with apharmaceutical agent at 37° C. for 2 hr. The cells were washed once withPBS and re-suspended in cell lysis buffer (60 mM Tris (pH 6.8), 2% SDS,10% glycerol, 5% betamercaptoethanol, 0.001% bromophenol blue) andultrasonicated, then used as a whole cell lysate for western blotting.

The whole cell lysate corresponding to the protein amount of 25 μg wassubjected to 7.5% SDS-polyacrylamide gel electrophoresis, and thentransferred onto a PVDF membrane. The membrane was blocked, andincubated with anti-phosphotyrosine mouse monoclonal antibody PY20(Transduction Laboratories, catalog No. P11120) in a 0.1% Tween 20 addedTris buffer, after which treated with HRP labeled anti-mouse secondaryantibody (DAKO, catalog No. P0447). The membrane was treated withchemiluminescent reagent ECL western blotting detection reagents(Amersham Pharmacia Biotech, catalog No. RPN2209) and thechemiluminescence was photographed with a luminoCCD camera. Thephotographing of the chemiluminescence and image analysis were performedusing Densitograph for Macintosh (ATTO, product type AE-6930).

The obtained phosphorylation signals were quantified and evaluated forphosphorylation inhibition by the pharmaceutical agent in % control,wherein the signal without addition of the compound was 100% control andthe background signal was 0%. The results are shown in the followingTable. TABLE 2 cellular Her2 tyrosine kinase inhibitory activitypharmaceutical % control % control agent (0.1 μM) (1 μM) PD 77 12

From the results of Examples 1, 2, it is recognized that PD shows aninhibitory activity on both Her2 and EGFR, namely, PD acts as a Her2and/or EGFR inhibitor.

Example 3 In Vivo Antitumor Effects

(Method)

Human pancreatic cancer HPAC (ATCC No. CRL-2119), human colorectalcancer LS174T (ATCC No. CL-188) and human lung cancer NCI-H520 (ATCC No.HTB-182) were purchased from ATCC. Human cervical cancer ME180 wassupplied by Cell Resource Center for Biomedical Research, the Instituteof Development, Aging and Cancer, Tohoku University (catalog No.TKG-0437, hen purchased from ATCC, ATCC No. HTB-33). Cultured humancancer cells suspended in PBS were implanted subcutaneously in the backof female Balb/c nude mice (Balb/cAJcl-nu mouse, CLEA Japan Inc.,5-week-old when received) at 5×10⁶/100 μl. When about 7 days later andthe average volume of the implanted tumors almost reached 100 mm³, themice were allotted by 4 mice per group such that the average tumorvolume of each group became equal.

For the tumor volume, the longer diameter and the shorter diameter weremeasured with calipers and calculated as [(shorter diameter)²×(longerdiameter/2)]=tumor volume [mm³]. A pharmaceutical agent was administeredby oral gavage once a day for 14 consecutively days from the day ofallottment, and the pharmaceutical agent was not administered to themice of the control group. The relative tumor growth rate was calculatedbased on the tumor volume on the administration start day for thepharmaceutical agent treatment group against the control group asrate 1. The antitumor effects are shown in % control. The % control wascalculated by the following formula.% control=[(average relative tumor growth rate of pharmaceutical agenttreatment group on the final day-1)/(average relative tumor growth rateof control group on the final day-1)]×100

The results are shown in the following Table. TABLE 3 Antitumor effectson human pancreatic cancer HPAC (both EGFR, Her2 positive) averagerelative pharmaceutical tumor growth agent dose mg/kg rate % controlcontrol — 4.6 100 PD 10 2.4 39 PD 30 1.4 11

TABLE 4 Antitumor effects on human cervical cancer ME180 (EGFR positive,Her2 negative) average relative pharmaceutical dose tumor growth agentmg/kg rate % control Control — 10.9 100 PD.2HCl 10 3.5 25 PD.2HCl 30 2.717

TABLE 5 Antitumor effects on human rectal cancer LS174T (EGFR negative,Her2 positive) average relative pharmaceutical dose tumor growth agentmg/kg rate % control control — 18.2 100 PD 10 10.7 56 PD 30 6.8 34

TABLE 6 Antitumor effects on human lung cancer NCI-H520 (both EGFR, Her2negative) average relative pharmaceutical dose tumor growth agent mg/kgrate % control Control — 8.7 100 PD.2HCl 10 8.3 95 PD.2HCl 30 7.7 87

From the results shown in above Tables, it is recognized that a Her2and/or EGFR inhibitor shows a growth suppressive effect on cancer cellsof (both EGFR, Her2 positive), (EGFR positive, Her2 negative) or (EGFRnegative, Her2 positive), namely, that a Her2 and/or EGFR inhibitor iseffective for the prophylaxis or treatment of a disease caused byoverexpression or activation of Her2 and/or EGFR.

Example 4 Confirmation of Expression of EGFR or Her2 by Western Blotting

(Method)

Human pancreatic cancer HPAC (ATCC No. CRL-2119), PANC1 (ATCC No.CRL-1469), human lymphoma Daudi (ATCC No. CCL-213) and human colorectalcancer LS174T (ATCC No. CL-188) were purchased from ATCC. human vulvarcancer A431 (catalog No. TKG-0182), human cervical cancer ME180 (catalogNo. TKG-0437), human tongue cancers HSC-3 (catalog No. TKG-0484) andHSC-4 (catalog No. TKG-0489) were provided by Cell Resource Center forBiomedical Research, the Institute of Development, Aging and Cancer,Tohoku University.

The cells were cultured in a 100 mm culture dish until they becamealmost confluent. The medium in the 100 mm culture dish were removed andwashed once with PBS. Then, about 0.6 mL of RIPA buffer (50 mM Tris (pH7.4), 150 mM sodium chloride, 1% NP-40, 0.25% deoxycholic acid, 1 mMEDTA and protease inhibitor cocktail) was added and the mixture wasstood on ice for 10 min. The cell lysate was recovered in a 1.5 mLcentrifugation tube and centrifuged at 10,000 rpm for 10 min withcooling. The supernatant was transferred to a different tube and usedfor western blotting.

The cell lysate corresponding to the protein amount of 25 μg wassubjected to 7.5% SDS-polyacrylamide electrophoresis, and thentransferred onto a PVDF membrane. The membrane was blocked, andincubated with a specific antibody against EGFR (Santa CruzBiotechnology, catalog No. sc-03) or a specific antibody against Her2(Upstate biotechnology, catalog No. 06-562) in a 0.1% Tween 20 addedTris buffer together, after which treated with HRP labeled anti-rabbitsecondary antibody (ICN Pharmaceuticals, catalog No. 55689) . Themembrane was treated with chemiluminescent reagent ECL western blottingdetection reagents (Amersham Pharmacia Biotech, catalog No. RPN2209) andthe chemiluminescence was photographed with a luminoCCD camera.

The images are shown in FIG. 1. In addition, explanation of each lane inFIG. 1 is shown in the following Table. TABLE 7 upper column lowercolumn EGFR Her2 lane cell line expression expression 1 HPAC positivepositive 2 PANC1 positive positive 3 ME180 positive negative 4 Daudinegative negative 5 A431 positive positive 6 LS174T negative positive 7HSC3 positive positive 8 HSC4 positive positive

Example 5 Confirmation of Expression of EGFR or Her2 by RT-PCR Method

(Method)

The test was performed according to Molecular Cloning., A LaboratoryManual Vol. 1, Second Edition. The detail is given in the following.

Total RNA was extracted from cancer cells with S.N.A.P.™ total RNAisolation kit (invitrogen, catalog No. 45-0472). 1 μg of the obtainedtotal RNA was used for reverse transcription reaction. The reversetranscription reaction was performed using 1st Strand cDNA Synthesis Kitfor RT-PCR (Roche, catalog No. 1 483 188).

As the primer used then was random primer p(dN)6. 1 μL of the obtainedcDNA was used to perform PCR. For detection of EGFR mRNA, HumanEpidermal growth factor receptor and/or GAPDH genes Dual-PCR kit (MaximBiotech, catalog No. DP-10065-G) attached primer was used as a primerand TaKaRa Ex Taq^(TMb) TAKARA SHUZO CO., LTD., catalog No. RR001A) wasused as an enzyme. The reaction conditions were 1 cycle of 96° C. 1minute, 30 cycles of 96° C. 1 minute and 58° C. 1 minute 30 seconds, and1 cycle of 72° C. 10 minutes. For detection of Her2 mRNA, RT-PCR primerset HUMAN erb-B2 (CLP, catalog No. 5254.H)attached primer was used as aprimer and TaKaRa Ex Taq™ (TAKARA SHUZO CO., LTD., catalog No. RR001A)was used as an enzyme. The reaction conditions were 1 cycle of 94° C. 5minutes and 60° C. 5 minutes, 40 cycles of 72° C. 2 minutes, 94° C. 1minute and 60° C. 1 minute, and 1 cycle of 72° C. 10 minutes. Theobtained PCR product was subjected to agarose electrophoresis forconfirmation of the expression.

From the results of the above-mentioned Examples 4, 5, it is appreciatedthat confirmation of the expression of Her2 and/or EGFR is achieved bywestern blotting or RT-PCR method.

From the results of the above-mentioned Examples 2, 4, moreover, it isappreciated that the test of Her2 or EGFR activation is possible. Usingsuch a test method, the expression or activity of Her2 and/or EGFR inpatients is tested, and when overexpression or activation of Her2 and/orEGFR are/is diagnosed, patient is evaluated as suffering from a diseasecaused by the expression or activation of Her2 and/or EGFR, and thepharmaceutical product of the present invention is administered.

INDUSTRIAL APPLICABILITY

As mentioned above, the inhibitor of the present invention is aneffective treatment method of cancer patients and also expected to be anagent for the prophylaxis and/or treatment of preventing transition fromhormone sensitive cancer to resistant cancer in prostate cancer andbreast cancer. Moreover, it is expected to an agent for the prophylaxisand/or treatment of angiogenesis associated with the growth of solidcancer and sarcoma, angiogenesis associated with cancer metastasis,angiogenesis associated with diabetic retinopathy, arteriosclerosis,psoriasis and the like.

This application is based on a patent application No. 162130/2002 filedin Japan, the contents of which are hereby incorporated by reference.

1. A Her2 and/or EGFR inhibitor to be administered to a subjectdetermined to show overexpression or activation of Her2 and/or EGFR as aresult of a diagnosis of the subject for the expression or activity ofHer2 and/or EGFR based on a test for detecting the expression oractivity of Her2 and/or EGFR.
 2. The inhibitor of claim 1 to beadministered to a subject determined to show activation of Her2 and/orEGFR as a result of a diagnosis of the subject for the activity of Her2and/or EGFR based on a test for detecting the activity of Her2 and/orEGFR.
 3. The inhibitor of claim 1 to be administered to a subjectdetermined to show overexpression or activation of Her2 and EGFR as aresult of a diagnosis of the subject for the expression or activity ofHer2 and EGFR based on a test for detecting the expression or activityof Her2 and EGFR.
 4. The inhibitor of claim 3 to be administered to asubject determined to show activation of Her2 and EGFR as a result of adiagnosis of the subject for the activity of Her2 and EGFR based on atest for detecting the activity of Her2 and EGFR.
 5. The inhibitor ofclaim 1, wherein the subject is a patient expected to suffer from adisease caused by overexpression or activation of Her2 and/or EGFR. 6.The inhibitor of claim 1, wherein the subject is a patient expected tosuffer from a disease caused by overexpression or activation of Her2 andEGFR.
 7. The inhibitor of any of claim 1, wherein the subject is ahuman.
 8. The inhibitor of claim 1, wherein the test for detecting theexpression or activity of Her2 and/or EGFR is an extracorporeal test. 9.The inhibitor of claim 1, wherein the test for detecting the expressionor activity of Her2 and EGFR is an extracorporeal test.
 10. Theinhibitor of claim 3, which is a mixture of a Her2 inhibitor and an EGFRinhibitor.
 11. The inhibitor of any one of claims 1 to 9, which is usedfor administering a Her2 inhibitor and/or an EGFR inhibitorsimultaneously, separately or at time intervals.
 12. The inhibitor ofclaim 8 or 9, wherein the extracorporeal test is an immunological methodusing an antibody, or a hybridization method using a nucleic acid and anucleic acid derivative.
 13. The inhibitor of claim 12, wherein theimmunological method using an antibody is selected from the groupconsisting of an enzyme-linked immunosorbent assay, an enzyme-linkedimmunoassay, a radioimmunoassay, an immunohistochemical method andwestern blotting.
 14. The inhibitor of claim 12, wherein thehybridization method using a nucleic acid and a nucleic acid derivativeis selected from the group consisting of an RT-PCR method, an ISHmethod, a FISH method, northern blotting and southern blotting method.15. The inhibitor of any one of claims 1 to 14, which is a substitutedheteroaromatic compound represented by the following formula (I)

wherein X is N or CH; Y is CR¹ and V is N; or Y is N and V is CR¹; or Yis CR¹ and V is CR²; or Y is CR² and V is CR¹; R¹ is C₁₋₄ alkyl, C₁₋₄alkoxy, CH₃SO₂CH₂CH₂NHCH₂—Ar— (wherein Ar is selected from phenyl,furan, thiophene, pyrrole and thiazole, each of which is optionallysubstituted by 1 or 2 halogens, C₁₋₄ alkyl or C₁₋₄ alkoxy on demand) or—C≡C—C(R⁶)(R⁷)(R⁸) (wherein R⁶, R⁷ and R⁸ are each independently ahydrogen atom, hydroxy, halogen, C₁₋₄ alkyl or C₁₋₄ alkoxy, or C₃₋₆cycloalkyl wherein the ring is optionally substituted by hydrogen atomor C₁₋₄ alkyl and optionally contains 1 or 2 hetero atoms selected fromO, S and N therein; R² is selected from the group consisting ofhydrogen, halogen, hydroxy, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylamino,di[C₁₋₄ alkyl]amino and —NHCO—R⁹ (wherein R⁹ is C₁₋₄ alkyl, C₁₋₄ alkoxy,C₂₋₄ alkenyl or C₂₋₄ alkynyl); U is phenyl, pyridyl, 3H-imidazolyl,indolyl, isoindolyl, indolinyl, isoindolinyl, 1H-indazolyl,2,3-dihydro-1H-indazolyl, 1H-benzimidazolyl,2,3-dihydro-1H-benzimidazolyl or 1H-benzotriazolyl group, each of whichis substituted by R³ group and optionally substituted on demand by atleast one R⁴ group selected independently; R³ is selected from the groupconsisting of benzyl, halo-, dihalo- and trihalobenzyl, benzoyl,pyridylmethyl, pyridylmethoxy, phenoxy, benzyloxy, halo-, dihalo- andtribenzyloxy and benzenesulfonyl; or R³ is trihalomethylbenzyl ortrihalomethylbenzyloxy; or R³ is a group of the above-mentioned formula(a) (wherein each R⁵ is independently selected from halogen, C₁₋₄ alkyland C₁₋₄ alkoxy; and n is 0-3); each R⁴ is independently hydroxy,halogen, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ alkoxy, amino,C₁₋₄ alkylamino, di[C₁₋₄ alkyl]amino, C₁₋₄ alkylthio, C₁₋₄alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ alkylcarbonyl, carboxy,carbamoyl, C₁₋₄ alkoxycarbonyl, C_(1-4 alkanoylamino, N-(C) ₁₋₄alkyl)carbamoyl, N,N-di(C₁₋₄ alkyl)carbamoyl, cyano, nitro ortrifluoromethyl, or a pharmaceutically acceptable salt thereof, ahydrate or solvate thereof, an optically active substance or a racematethereof, or a mixture of diastereomers thereof.
 16. The inhibitor ofclaim 15, which is(4-(3-fluorobenzyloxy)-phenyl)-(6-(5-((2-methanesulfonyl-ethylamino)methyl)-furan-2-yl)-pyrido[3,4-d]pyrimidin-4-yl)-amine;(4-benzyloxyphenyl)-(6-(5-((2-methanesulfonyl-ethylamino)-methyl)-furan-2-yl)-quinazolin-4-yl)-amine;N-{4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-}2-furyl]-4-quinazolinamine;N-[4-(benzyloxy)phenyl]-7-methoxy-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine;N-(1-benzyl-1H-indazol-5-yl)-7-methoxy-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine; N-{3-fluoro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine;N-[1-(3-fluorobenzyl)-1H-indazol-5-yl]-6-[2-({[2-(methylsulfonyl)ethyl]amino}methyl)-1,3-thiazol-4-yl]-4-quinazolinamine;6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-N-[4-(phenylsulfonyl)phenyl]-4-quinazolinamine;N-{3-fluoro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[2-({[2-(methylsulfonyl)ethyl]amino}methyl)-1,3-thiazol-4-yl]-4-quinazolinamine;N-(1-benzyl-1H-indazol-5-yl)-6-[2-({[2-(methylsulfonyl)ethyl]amino}methyl)-1,3-thiazol-4-yl]-4-quinazolinamine;N-(3-fluoro-4-benzyloxyphenyl)-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-4-furyl]-4-quinazolinamine;N-(3-chloro-4-benzyloxyphenyl)-6-[2-({[2-(methylsulfonyl)ethyl]amino}methyl)-4-furyl]-4-quinazolinamine;N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine;N-(1-benzyl-1H-indazol-5-yl)-7-fluoro-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine;N-(3-trifluoromethyl-4-benzyloxyphenyl)-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-4-furyl]-4-quinazolinamine;N-[4-(3-chloro-4-fluorophenyl)amino-7-[3-(4-morpholinyl)propoxy]quinazolin-6-yl]acrylamide;N-{4-[(3-chloro-4-fluorophenyl)amino]-7-[3-methyl-3-(4-methyl-1-piperazinyl)-1-butynyl]-6-quinazolinyl}acrylamide;orN-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine,or a pharmaceutically acceptable salt thereof, a hydrate or a solvatethereof, an optically active substance or a racemate thereof, or amixture of diastereomers thereof.
 17. The inhibitor of claim 15, whichisN-[4-(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]quinazolin-6-yl]acrylamide,orN-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamineor a pharmaceutically acceptable salt thereof, a hydrate or a solvatethereof, an optically active substance or a racemate thereof, or amixture of diastereomers thereof.
 18. A pharmaceutical compositioncomprising an inhibitor of any one of claims 1 to 17 as an activeingredient and a pharmaceutically acceptable carrier.
 19. Thepharmaceutical composition of claim 18, which is an agent for theprophylaxis and/or treatment of a disease caused by overexpression oractivation of Her2 and/or EGFR.
 20. The pharmaceutical composition ofclaim 19, wherein the disease caused by the overexpression or activationof Her2 and/or EGFR is cancer, angiogenesis associated with the growthof cancer or sarcoma, angiogenesis associated with cancer metastasis,angiogenesis associated with diabetic retinopathy, arteriosclerosis orpsoriasis.
 21. An agent for the prophylaxis and/or treatment of adisease caused by overexpression or activation of Her2 and/or EGFR,which is to be administered to a subject determined to showoverexpression or activation of Her2 and/or EGFR as a result of adiagnosis of the subject for the expression or activity of Her2 and/orEGFR based on a test for detecting the expression or activity of Her2and/or EGFR.
 22. The agent of claim 21, wherein the disease caused byoverexpression or activation of Her2 and/or EGFR is cancer, angiogenesisassociated with the growth of cancer or sarcoma, angiogenesis associatedwith cancer metastasis, angiogenesis associated with diabeticretinopathy, arteriosclerosis or psoriasis.
 23. A method for theprophylaxis and/or treatment of a disease caused by overexpression oractivation of Her2 and/or EGFR, which comprises administering aneffective dose of a Her2 and/or an EGFR inhibitor to a subjectdetermined to show overexpression or activation of Her2 and/or EGFR as aresult of a diagnosis of the subject for the expression or activity ofHer2 and/or EGFR based on a test for detecting the expression oractivity of Her2 and/or EGFR.
 24. The method of claim 23, wherein thedisease caused by overexpression or activation of Her2 and/or EGFR iscancer, angiogenesis associated with the growth of cancer or sarcoma,angiogenesis associated with cancer metastasis, angiogenesis associatedwith diabetic retinopathy, arteriosclerosis or psoriasis.
 25. Acommercial package comprising the pharmaceutical composition of any oneof claims 18 to 20 and a written matter associated therewith, thewritten matter stating that the pharmaceutical composition can or shouldbe used for the prophylaxis and/or treatment of a disease caused byoverexpression or activation of Her2 and/or EGFR.
 26. The commercialpackage of claim 25, wherein the disease caused by overexpression oractivation of Her2 and/or EGFR is cancer, angiogenesis associated withthe growth of cancer or sarcoma, angiogenesis associated with cancermetastasis, angiogenesis associated with diabetic retinopathy,arteriosclerosis or psoriasis.